The invention relates to a method for local analysis and distribution analysis (“imaging” or “mapping”) for the quantitative determination of element concentrations in substrates, in particular in thin tissue sections, of individual cells or cell organelles, and to the in situ characterization of sample surfaces (topography) prior to and after chemical analysis with lateral resolution in the micrometer to nanometer range. The invention further relates to an apparatus that is suitable for carrying out the aforementioned methods.
In element mass spectrometry for direct ablation of the sample material to be examined, various methods, for example using focused laser beams, are employed as analysis methods for the quantitative determination of lateral element distributions with a spatial resolution in the μm range, and for determining trace elements to the ng/g and sub-ng/g concentration range. For example, the method of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is known, which is employed for trace and isotope analyses, as well as for surface and microlocal analyses on solid sample materials. The disadvantage of this method, however, is that it is not able to determine element distributions and concentrations in thin tissue sections (mapping or imaging analysis) with high lateral resolving power, and notably in the lower microscale and nanoscale range below 5 μm, as is required, for example, for the analysis of individual cells or cell organelles.
In analytics, lasers are increasingly employed, for example the Nd:YAG laser, having a wavelength in the UV range (λ-266 nm or 213 nm). These lasers are frequently used for sample introduction, combined with sensitive ICP mass spectrometers. At present, Nd:YAG laser systems having a laser spot diameter of several μm to several hundred μm are commercially available for microlocal analysis (for example, LSX 213, 500 CETAC Technologies, Ohama, USA, or UP 213, 266 New WAVE Research, Fremont, USA). It is perceived to be a disadvantage that, in general, it is not possible to use such commercial laser ablation systems to directly ablate biological matrices at a lateral resolving power in the lower μm range with high efficiency. The limiting factor here is the diffraction limit below which a laser can generally not go. This means that the minimum possible resolution limit is in the range of the wavelength of the laser. Spatially resolved analyses below 1 μm are therefore generally not possible at all.
Many medical-molecular biological questions, for example regarding the distribution analysis of metals in biological samples (such as in brain sections), however, require that the lateral resolving power of the analysis method used be in the lower micrometer, or several hundred nm range, or as low as the lower nm range. Such spatial resolution could mean that direct nanolocal analytics would be feasible on individual cell organelles. Moreover, many questions demand quantitative information about the metal distribution of diseased tissue sections compared to healthy tissue sections.
Focused IR and UV laser beams are used in commercial instruments (by means of known laser-assisted micro dissection, LMD) to cut out an exactly defined area from a tissue. Laser-assisted micro dissection (LMD), which allows for specific molecular-genetic examination of minimal quantities of a specific tissue, also allows isolated examination of select regions of living individual cells. Such powerful LMD systems having spatial resolution in the lower micrometer range, or at times in the sub-micrometer range, are employed today for isolating and analyzing individual cells or cell areas, for example in the biopsy of individual cancer cells compared to surrounding healthy control cells, or small tissue pieces or DNA strands, for routine research tasks in medicine (pathology), as well as in molecular biology and cell biology. Existing commercial systems offer excellent options for microscopic observation of the sample surfaces and very precise cut-outs of tissue pieces by means of a highly focused laser beam. When using special laser optics, this beam achieves high spatial resolution, as small as the sub-micrometer range (down to 0.5 μm). As differs from laser ablation systems—where the ablation of the sample material is controlled by a defined movement of the specimen stage—the laser beam is moved in a defined manner over the sample surface by means of sophisticated optics having a precision of approximately 0.07 μm. The cut-out tissue samples are subsequently supplied to further offline biomolecular mass spectrometric analysis, typically following tryptic digestion (cell lysis). Quantitative element analysis using LMD online, however, is usually not possible. Compared to commercially available laser ablation systems, such a LMD system has a number of outstanding advantages, such as improved spatial resolution by approximately one order of magnitude, significantly improved microscopic resolution and, in conjunction with special staining techniques (immunostaining) and the use of highly specialized software packages, the detection of special cells (for example, of cancer cells in stained tissue sections).